The goal of this proposal is to determine if a simple blood-based assay we have developed for unbound free fatty acids (FFAu), allows for an early and accurate diagnosis of ischemic stroke. Although reperfusion therapy using t-PA is an effective treatment for ischemic stroke it has been adopted slowly, in part because it must be administered within 3 hours of an ischemic stroke. The rapid and accurate diagnosis of stroke is a major barrier to the effective implementation of thrombolytic reperfusion therapy. What is needed is a simple test, for example one that would assay for a stroke marker in a small quantity of blood, with high sensitivity and specificity at the earliest times after symptoms are apparent. The fluorescent (ADIFAB) method we have developed requires only a single step, uses less than 0.1 ml of blood, is complete in seconds, and can be done with a hand-held device. Our preliminary results indicate that plasma FFAu levels can detect myocardial ischemia within minutes of the insult and evidence from animal studies suggest the same for cerebral ischemia. We will therefore use blood specimens collected by the UCSD Stroke Team to determine, the time course of FFAu in stroke and stroke mimics, whether FFAu levels can be used to monitor the course of t-PA therapy, and if FFAu levels can be used to help group patients for treatment. Preliminary results using this assay for specimens from the TIMI II trial indicate that FFAu levels, at presentation (before t-PA administration), predict increased risk of death and severe hemorrhagic events. We will therefore determine if similar correlations exist in stroke code patients and if therapies designed to reduce FFAu levels improve outcome in animal models. Finally, we will undertake studies to determine the origin for the observed increased FFAu levels, because better understanding of the molecular mechanisms of this phenomenon, may lead to novel diagnostic and therapeutic strategies for ischemic stroke.